Since the confocal core facility opened in 1998, usage has steadily and rapidly increased. Over 259 users from 30 different CCR laboratories or branches used the facility this year. Fifty nine of these were new users that required extensive confocal training and supervision. Among the techniques now in use within our CCR confocal core facility are methods to visualize live cells in three dimensions over time, including the tracking of cell motion in three-dimensional tissue, in vivo imaging of differentiated embryonic stem cells in 3D, spontaneous fusion and asymmetric division of cancer stem cells, monitoring membrane fluidity of MDR cells using FRAP, and time lapse imaging of live cells expressing GFP-PKC isoforms to determine mode of drug action. Examples of projects using 2-photon technology include whole live lung imaging with 2-photon to determine metastatic potential of GFP expressing tumor cells and Second harmonic generation (SHG) imaging using the 2-photon laser in whole liver to determine morphological differences in collagen between control and Met Knockout mice. Another exciting area of expertise includes methods of in vivo biochemistry in which light microscopy is being used to assess whether two proteins physically interact at a certain time and place within a cell using Fluorescence Resonance Energy Transfer (FRET) technology as well as the use of FRET to measure drug potencies within the context of cell type. The Zeiss LSM 710 provides Fluorescence Lifetime Imaging (FLIM) with a Becker and Hickl detector and software analysis package. The new 32 channel detector on the Zeiss 710 system has also greatly improved multispectral imaging. The new Zeiss LSM 780 with GaAsP allows acquisition of up to 10 dyes and allows photon counting and Fluorescence Correlation Spectroscopy (FCS). Several projects conducted over the last few years have required specialized imaging techniques and expertise from the core. Both 1 photon and 2 photon FLIM is being employed on the Zeiss 710 by Dr. Petr Kalab to study Ran GTPase function in mitotic spindle assembly. The RanGTP concentration gradient surrounding mitotic chromosomes induces the formation of a gradient of spindle assembly factors (SAFs) activated by RanGTP-induced release from inhibitory complexes of SAFs with importin b. FLIM is used to detect the signal of a FRET-based biosensor that reports its RanGTP-induced liberation from importin b. We have continued a project with Dr. Arnulfo Mendoza using second harmonic generation and autofluorescence with the 2-photon laser to image live lung sections and evaluate structural/morphological differences after tumor cell expansion. A collaborative project with Dr. Benu Das continues to use FRAP analysis to investigate the DNA damage response, specifically the serine 81 phosphorylation of TDP1, a DNA repair enzyme. TDP1 promotes binding to XRCC1 and enhances the mobilization of TDP1 to the sites of damage together with XRCC1. Another ongoing project involves colocalization analysis of CARP-2, a negative regulator of TNF-induced NF-kB activation, and cellular organelles. New projects for confocal imaging this year included Dr. Luowei Lis project on a Euphorbia peplus extract (PEP005) which is in clinical trials for eradicating basal cell carcinoma. Confocal imaging and 3D surface rendering helped determine the effect of this compound as well as other phorbol esters on the vasculature of skin tumors. A collaboration with Dr. Chava Kimchi-Sarfaty, FDA, used confocal imaging to validate a new flow cytometry method for the detection of intracellular levels of ADAMTS13, a secreted metalloprotease that cleaves von Willebrand Factor multimers and maintains proper homeostasis. Deficiency in ADAMTS13 can lead to the disorder known as thrombotic thrombocytopenic purpura (TTP). Another new project is being conducted with Dr. Binwu Tang to image cancer stem cells expressing SOX2 and OCT4 and to monitor the TGF beta function in these cells by co-transfecting GFP constructs for SOX2/OCT4 and a construct with a TGF beta response element and RFP expression. A collaboration with Dr. Julio Valencia is using confocal imaging to help define the molecular mechanisms regulating the dual promoter region of CDK2 and Pmel17 which will improve targeted therapies for melanomas. He has shown the importance of regulating a dual promoter region shared by CDK2 and Pmel17, two genes critical to melanocytic cell growth and differentiation, respectively. Transcriptional, biological and chemical approaches were used to further clarify these processes and identified a mechanism by which two p53-regulated genes(NFYA and FHL2) play key roles in the regulation of melanoma cell growth, differentiation and carcinogenesis.